An MLF1 deficiency causes more immature AML in Trib1 mice. (A) Schematic representation of the Mlf1 gene targeting strategy. The coding exon 1 is shown as a filled box; an open box denotes a noncoding portion. The probe used for a Southern blot analysis is shown as a gray box. Primers a, b, and c used for genomic PCR are indicated as arrows with the amplified direction. Neo, the neomycin phosphotransferase gene; TK, thymidine kinase gene. A restriction site of HindIII is shown as a single letter, H. (B) A Southern blot analysis of the targeted Mlf1 locus (left panel). Genomic DNA was extracted from ES cell clones, digested with HindIII, and hybridized with the probe. The sizes of WT (7.4 kb) and disrupted (3.2 kb) alleles are indicated. A PCR analysis of genomic DNA isolated from WT and knockout mouse tails (right panel). The positions of WT (primers a and b, 409 bp) and mutant (KO, primers b and c, 819 bp) amplification products are indicated. (C) Myeloid leukemia–free survival curves of mice transplanted with Trib1-transduced Mlf1^−/− BM cells (Mlf1^−/− Trib1 mice) compared with Trib1-transduced WT BM cells (WT Trib1 mice) and MigR1 control vector-transduced Mlf1^−/− BM cells (Mlf1^−/− MigR1 mice). Results are derived from 5 independent transfer experiments. The P value between WT Trib1 and Mlf1^−/− Trib1 mice was calculated with the log-rank test. P = .14. (D) May-Grünwald Giemsa–stained PB smears and cytospins of BM cells from WT Trib1 (left panel), Mlf1^−/− Trib1 (middle panel), and Mlf1^−/− MigR1 (right panel) mice. Original magnification ×400. (E) A FACS analysis of BM and SP cells for immature (Mac-1⁺Gr-1^lo) and differentiated (Mac-1⁺Gr-1^hi) granulocytes. The population of GFP-positive cells in BM and SP is shown in the upper panels. (F) A detailed FACS analysis of GFP-positive BM cells for the lineage negative cell (Lin⁻: CD3⁻B220⁻TER-119⁻Mac-1⁻Gr-1⁻), stem cell (LSK cells), and multipotent progenitor (CMP: Lin⁻c-Kit⁺Sca-1⁻FcγR^loCD34⁺; GMP: Lin⁻c-Kit⁺Sca-1⁻FcγR^hiCD34⁺; MEP: Lin⁻c-Kit⁺Sca-1⁻FcγR^loCD34⁻). (G) A detailed FACS analysis of GFP-positive BM cells for the lineage negative cell excluding Mac-1 and Gr-1 (Lin*⁻: CD3⁻B220⁻TER-119⁻), committed myeloid progenitor (Lin*⁻Sca-1⁻c-Kit⁺Mac-1⁺), and differentiated myeloid cell (Lin*⁻Sca-1⁻c-Kit^lo/−Mac-1⁺). (H) Total RNA extracted from GFP-positive BM cells was analyzed by semi-qRT-PCR using a pair of primers specific to COP1, Trib1, and β-actin.